Field of the Invention
The present invention pertains to a method of extending life span in organisms and delays the onset and many of the complications associated with age-related diseases, including cancer. More particularly, the invention relates to the administration of a chemical agent to upregulate and downregulate the expression (i.e. gene activation) of the same beneficial genes that are activated in caloric restriction. The genes are activated by mimicking the same intracellular conditions as are seen in caloric restriction, but without the need to reduce caloric intake. Compositions and methods to prolong life and protect an organism from age-related diseases are likewise provided.
Description of the Related Art
Many attempts have been made to extend life span in single cell organisms and multi-cellular animals. These attempts have included various nutritionally-based interventions, vitamin supplements, antioxidant supplements, exercise, hormonal, pharmaceutical and other paradigms (Lane, M. et al. Nutritional Modulation of aging in nonhuman primates, 1999 The Journal of Nutrition, Health & Aging, Vol. 3, No. 2 pp 69-76). While these attempts sometimes result in better health, in the last 70 years, only activation of beneficial genes has caused an increase in lifespan. Three methods of beneficial gene activation have been proven to extend mean and maximal lifespan: 1) gene activation by calorie restriction (CR); 2) certain types of animals receiving genetic engineering (the artificial addition or deletion of genes); and 3) the use of chemicals that activate the Sir2 gene by lowering the Michaelis constants, Km, of the Sir-2 enzymes for the co-substrate NAD+[24]. CR is the limitation of total calories derived from carbohydrates, fats, or proteins to a level 25% to 60% below that of control animals fed ad libitum (Koubova et al, How does calorie restriction work? 2003 Genes & Development. Vol. 17 pp 212-221). Success in extending lifespan with gene activation by CR includes a wide range of different organisms including yeast, rotifers, guppies, spiders, fruit flies, hamsters, rats, mice and it is now indicated at extending lifespan in primates (Lane et al.; Koubova et al.; Lane et al, Short-term calorie restriction improves disease-related markers in older male rhesus monkeys (Macaca mulatta) 1999 Mechanisms of Ageing and Development Vol. 112 pp 185-196). Success in extending lifespan with genetic engineering has been successful in yeast, worms, fruit flies and mice (Hekimi, S. et al, Genetics and the Specificity of the Aging Process, Science. 2003 Feb. 28; 299(5611):1351-4. Review; Guarente, L. SIR2 and aging—the exception that proves the rule, Trends Genet. 2001 July; 17(7):391-2; Tissenbaum, H et al. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans, 2001 Nature Vol 410 pp 227-230; Lin, S et al, Requirement of NAD and SIR2 for Life-Span Extension by Calorie Restriction in Saccharomyces cerevisiae 2000 Science Vol. 289 pp 294-297; Lin, S et al, Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration 2002 Nature, Vol. 110 pp 244-248; Guarente, L. Mutant mice live longer, 1999 Nature Vol. 402 pp 243-245). Success in extending lifespan with chemicals that lower the Michaelis constant of the Sir-2 enzymes for NAD has been shown in yeast and worms (Howitz et al., Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan, Nature 425: 191-196; Wood et al., Sirtuin activators mimic caloric restriction and delay ageing in metazoans, Nature, Volume 430, 5 Aug. 2004. In all cases, expansion of lifespan required the activation of beneficial genes.
It is significant that CR works on such a wide range of organisms, from the single celled to very complex (including primates). The wide range of success of CR indicates that the process of life extension is based on the effects within the individual cells of the organisms, and that the process allowing life span extension is preserved across species. In rodents, the extension in life span can approach 50% (Koubova et al.). This lifespan comes at a price, however, as the organism needs to be fed at least 25% less calories than it would normally consume.
The benefits of CR are numerous. In addition to lifespan extension, the onsets of aging-related diseases are also delayed, leading to a healthier organism for a longer time. In mammals, CR delays all kidney disease, autoimmune disease, and diabetes. CR reduces age associated neuron loss in mouse models of Parkinson's disease and Alzheimer's disease (Koubova et al.). It is also noted that even moderate CR lowers cancer risk in mammals (Mai, V. Even Moderate Caloric Restriction Lowers Cancer Risk in Mice, Experimental Biology Conference 2002 Apr. 23 meeting). Additionally, CR mammals have been observed to have less body fat (Picard, et al., Sirt1 promotes fat mobilization in white adipocytes by repressing ppar gama, Nature, Vol. 429, 17 Jun. 2004.). CR has been shown to enhance the repair of DNA in skin and other tissues after exposure to ultraviolet light (Lipman et al, “The influence of dietary restriction on DNA repair in rodents: a preliminary study”, Mech Ageing Dev 1989: 48: 135-43; Weraarchakul et al, “The effect of aging and dietary restriction on DNA repair”, Exp Cell Res 1989; 181: 197-204; Licastro et al, “Effect of dietary restriction upon the age-associated decline of lymphocyte DNA repair activity in mice”, Age 1988: 11: 48-52; Srivastava et al, “Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: Effects of caloric restriction”, Biochem Biophys Res Commun 1992: 182: 712-21; Tilley et al, “Enhanced unscheduled DNA synthesis by secondary cultures of lung cells established from calorically restricted aged rats”, Mech Ageing Dev 1992: 63” 165-76). DNA repair is critical for skin repair and to prevent skin aging. It also reduces skin cancer incidence. Studies of humans undergoing CR for 3 to 15 years have shown reduced risk for atherosclerosis along with reductions in fasting glucose, fasting insulin, Hs-CRP levels, systolic and diastolic blood pressure, triglycerides, total cholesterol, and LDL cholesterol as compared to equivalent age-matched controls (Fontana, et al, “Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans, PNAS, Apr. 27, 2004, Vol. 101, no. 17, pp 6659-6663).
The benefits of CR are not due to dietary antioxidants, as single agents or combinations of antioxidants do not produce an increase in lifespan or delay tumorigenesis and other age related disease. Instead, CR works due to signaling changes that activate gene expression that reduce cellular proliferation or increase apoptosis. Multiple genes involved in the electron transport chain, immune response, protein turnover and protein synthesis are changed in CR (Lee, et al., The impact of α-Lipoic Acid, Coenzyme Q10, and Caloric Restriction on Life Span and Gene Expression Patterns in Mice, Free Radical Biology & Medicine, Vol. 36, No. 8, pp. 1043-1057, 2004). Masternak et al shows that genes related to insulin and insulin growth factor 1 (IGF1) are altered including PPARα, a gene suggested to play an important role in metabolic control and the accumulation and preservation of fat storage cells. (Masternak, et. al., Divergent Effects of Caloric Restriction on Gene Expression in Normal and Long-Lived Mice, Journal of Genontology, 2004, Vol. 59A, No. 8, 784-788). The activity of FOXO genes have also been shown to change under caloric restriction (Daitolku, et al., Silent information regulator 2 potentates Foxo1-mediated transcription through its deacetylase activity, PNAS, Jul. 6, 2004).
Within the last decade, it has been determined that the Silenced Information Regulator 2 (Sir2) gene in yeast and worms (Sir2.1 in worms, SIRT1 in humans) is also one of the genes that regulates lifespan and is activated in CR. Mutant worms and yeast with extra copies of Sir2 or Sir2.1 live longer, while mutations in the Sir2 gene severely reduce lifespan. See, e.g. Tissenbaum et al. Other animals contain similar genes or homologues to the Sir2 gene, including humans (the SIRT1 gene). CR creates a set of conditions in the cell that signals the activation of beneficial genes to lengthen lifespan and delay the onset of age-related disease. The activation of Sir2 by CR is one pathway to increased lifespan. CR also stimulates other genes that increase lifespan independent of Sir2 in a parallel pathway. Kaeberlein et al, “Sir2-Independent Life Span Extension by Calorie Restriction in Yeast” 2004, PloS Biology: 2: 9: e296: 1381-1387.
It has been shown that activation of Sir2 can activate or silence other genes and proteins, including FOXO type genes. Also, the activation of the Sir2 gene (SIRT1 in humans) normally turned on in CR blunted the protein PPAR gamma that activated fat-storage genes, so that fat cells would shed fat and prevented cells from differentiating into fat cells. See, e.g. Picard et al. supra. This would explain the low amounts of fat seen in mammals under CR.
Lin et al. determined that the internal cellular signaling condition generated by CR to activate beneficial genes is the increase in NAD+/NADH (oxidized and reduced nicotinamide adenine dinucleotide) ratios within the cell as compared to non-CR conditions. Lin, S. et al, Calorie restriction extends yeast life span by lowering the level of NADH. 2004 Genes & Development Vol. 18 pp 12-16. Lin also noted that NAD+ levels in cells remain constant between CR and non-CR conditions, while the reduced form of NAD+, NADH, is significantly lowered in CR (up to 50%), which allows activation of at least one beneficial gene, the Sir2 type gene. High levels of NADH are an inhibitor of the Sir2 gene.
Lin's study showed at least one of the intracellular requirements for signaling the activation of beneficial genes resulting in increased longevity and health benefits found during CR. The study used recombinant genetic modifications to achieve the increase the ratio of NAD+/NADH, (without the restriction in calories) and thereby “mimic” caloric restriction results of increased lifespan and general improvement in health. The important characteristic shown was that calories did not have to be reduced, but rather that beneficial genes need to be activated within the individual cells in order to achieve the same benefits of CR.
In other studies by Horitz, Wood and Lamming, researchers have discovered an alternate pathway for increasing life span that is distinct from CR and genetic engineering to increase the NAD+/NADH ratio to stimulate at least one beneficial gene. Instead of inserting genes to modify the NAD+/NADH ratio or to add additional copies of a beneficial gene by genetic engineering, they instead used chemical agents to lower the substrate-binding affinity between NAD+ and Sir2 allowing the Sir2 (SIRT1 in humans) to activate more readily. Howitz et al., Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan, Nature 425: 191-196; Wood et al., Sirtuin activators mimic caloric restriction and delay ageing in metazoans, Nature, Volume 430, 5 Aug. 2004; Lamming et al., Small molecules that regulate lifespan: evidence for xenohormesis, Molecular Microbiology, 2004, Vol. 53(4), 1003-1009. The chemical agents discovered are polyphenols and include the compound resveratrol. The polyphenols are found in plants, but are not part of the natural chemical makeup of mammals including humans. The polyphenols are very specific in the activation of the Sir2 gene and its homologues. The Sir2 gene extends lifespan, but does not activate all of the beneficial genes activated by CR. As a result of this, the Sir2 activating polyphenols produce a lower increase in lifespan extension than does CR. Kaeberlein et al, “Sir2-Independent Life Span Extension by Calorie Restriction in Yeast” 2004, PloS Biology: 2: 9: e296: 1381-1387.
Changing the inter-cellular binding potential with chemical agents or using genetic engineering to increase lifespan, reduce fat accumulation, and delay cancer and age related disease and improve overall health is a marvelous achievement. As a caution, however, genetic engineering is hardly a well-understood field, and is unlikely to help increase the lifespan of humans any time in the near future. Using chemical agents to lower the binding affinity of certain enzymes in order to stimulate Sir2 or Sirt1 (in humans) is also an uncertain path, as there is no long-term determination of risks. Additionally, the Sir2 or Sirt1 gene is only one of the genes that can be activated to increase lifespan, and produces a modest increase, whereas activation of more beneficial genes can result in longer increases in in lifespan. Finally, what if the application of the foreign chemicals such as Resveratrol cause harm in some isolated area of the human body?
The only long-term studies performed to extend lifespan, reduce body fat and delay cancer and other age-related conditions focused on actual caloric restriction. The studies, done since the 1930's, have shown the many benefits of caloric restriction, with the only noted potential disadvantages being that organisms took longer before they were of age to reproduce, and the organisms tended to be smaller than non-calorie restricted organisms.
We have been taught that the intercellular conditions seen in CR to activate beneficial genes include an increase in the NAD+/NADH ratio, which acts as a switching mechanism for the cell. To lower overall risk, it would be better to stimulate the same set of beneficial genes seen in CR by using the identical signaling method for the genes involved with CR. It would be beneficial to activate other life-extending genes in addition to or besides the Sir2 gene. Moreover, it would be of great benefit to find chemical agents that increase the NAD+/NADH ratio. Chemical agents that increase NAD+/NADH could provide a proven safe pathway (70 years of research) for lifespan expansion and the delay in the onset of age-related diseases. It would also be beneficial if the activation agent to increase the NAD+/NADH ratio was a chemical that is already found in mammals including humans, rather than introducing foreign compounds with unknown long-term results.
Due to the wide variety of chemical reactions available to the cell, each cell reacts in a manner to conserve the NAD+/NADH ratio. It is, in effect, a buffered response. It is especially difficult to increase the ratio. However, ethanol can decrease the NAD+/NADH ratio, which results in higher triglycerides and “fatty liver” disease.
Finding a compound to increase the NAD+/NADH ratio to activate beneficial genes is not trivial. One reason for this is due to the difficulty in directly measuring the NAD+/NADH ratio with current technology. Instead of measuring NAD+/NADH directly, the ratio is inferred indirectly by the measurement of the pyruvate/lactate ratio. Typically, when the amount of pyruvate to lactate increases, NAD+/NADH increases.
Thus, one method of increasing the NAD+/NADH ratio in the cells would be to increase the amount of pyruvate into the cell. In gluconeogenesis, pyruvate can be converted to glucose and converts a NADH to NAD+, which will increase the NAD+/NADH ratio. Also, under anaerobic conditions, pyruvate is converted to lactate by the enzyme lactate dehydrogenase. The conversion of pyruvate to lactate under anaerobic conditions again converts a NADH to NAD+. There are reports of an increase in the NAD+/NADH ratio with the injection of pyruvate into rats. Work done by Ido on the study of blood flow in the retina and visual cortex show that NADH levels in the cytosol can be dropped by 50%, doubling the NAD+/NADH ratio. Ido, et al, NADH augments blood flow in physiologically activated retina and visual cortex, PNAS, Jan. 13, 2004, Vol. 101, no. 2 pp 653-658. Despite this reported temporary change in the ratio, no extension of lifespan occurs with pyruvate because pyruvate also penetrates the inner mitochondrial membrane and preferentially engages in lowering the NAD+/NADH ratio through the Citric Acid Cycle. The ratio, temporarily raised by pyruvate, is then lowered when the pyruvate is processed through the Citric Acid Cycle. The typical cell buffers against increases in the NAD+/NADH ratio.
Anderson, et al. also had difficulty in using chemical agents to increase the NAD+/NADH ratio and activate beneficial genes. Anderson used acetaldehyde, known to reduce NADH in cells, but did not see any increase in the activity of beneficial genes. There is also some debate that changing the NAD+/NADH ratio will activate beneficial genes at all. Based on his work, Anderson teaches, “variations in NADH are unlikely to affect the activity of Sir2 or SIRT1” (beneficial genes) Anderson et al., Yeast Life-Span Extension by Calorie Restriction Is Independent of NAD Fluct . . . , Science 2003 302: 2124-2126.
There is a current need to create intracellular conditions similar to CR (i.e. increase of the NAD+/NADH ratio) with a Caloric Restriction “mimic” chemical that would allow beneficial genes to be implemented. Thus, the benefits of increased lifespan, lower cancer rates, lower body fat content and the delay in age-related disease without the heavy restrictions of diet imposed by CR or by genetic modification of the individual organism can be realized. The preference would be to have the chemical agents be currently part of human metabolism. The present invention provides such a chemical and method for the novel activation of beneficial genes.